The long-term objective of this proposal is to gain insight into the molecular mechanisms of cell apoptosis in response to microtubule disruption during cancer treatments. Microtubule disruption agents such as vinblastine, vincristine, and paclitaxel represent a growing family of anti-cancer drugs. They can effectively inhibit growth and induce apoptosis in a large variety of cancer cells, regardless of their p53 status. Little is known, however, about their mechanisms in inducing cell apoptosis. We have recently demonstrated, using a somatic knockout approach in DT40 cells, that MEKK1 is essential for JNK activation and cell apoptosis in response to microtubule or actin disruption. Our studies also indicated that this apoptotic process is independent of cell cycle arrest, but requires new protein synthesis. Through recent microarray analysis, we identified caspase 3 as a potential MEKK1-dependent apoptotic gene that is upregulated during microtubule disruption. In this proposal, we will test our hypothesis that cell apoptosis in response to microtubule or actin disruption proceeds through the MEKK1/JNK signal transduction pathway to upregulate proapoptotic genes such as caspase 3. Specific Aim 1 is focused on the molecular basis for the activation of the MEKK1. We will take structural and functional approaches, using MEKK1 -/- DT40 cells, to determine domains of MEKK1 required for JNK activation and apoptosis in response to microtubule and/or actin disruption, and the signaling specificity between MEKK1 and other MEKK family members. In Specific Aim 2, we will define the MEKK1-dependent apoptotic pathway by studying the role of SEK/JNK pathway and the upregulation of caspase 3 in microtubule disruption-induced apoptosis. In Specific Aim 3, we wilI determine the role of the MEKK1-dependent signaling pathway in the treatment of human cancer cells. We will measure the correlation between the drug sensitivity and the MEKK1 kinase activity, and determine whether it is possible to sensitize otherwise resistant human cancer cells to the treatment of microtubule disruption agents by increasing the activity of the MEKK1-dependent apoptotic pathway. We believe that our study will not only elucidate the molecular mechanisms of a large family of microtubule and actin disruption agents used in chemotherapy, but also will provide critical information for future design of more effective and specific anti-cancer drugs.